Project Summary Atherosclerosis is a significant cause of morbidity and mortality in the world, contributing to diseases such as coronary artery disease, peripheral arterial disease, and stroke. It is known that atherosclerosis has a focal propensity to areas of disturbed flow (d-flow) while areas of steady laminar flow (s-flow) are relatively protected. Endothelial cell (EC) activation by d-flow is an early step in atherosclerosis, and it is known that ECs exposed to d-flow show higher levels of inflammation, proliferation, apoptosis, and senescence. We have recently shown that ZBTB46 is a flow regulated transcription factor in ECs, is down-regulated by d-flow, and that its overexpression in ECs inhibits proliferation. While the role of ZBTB46 in atherosclerosis is not known, our preliminary studies suggest that ZBTB46 regulates a number of genes in ECs potentially involved in atherosclerosis, including P21 and SFRP2. We propose to test the overall hypothesis that decreased ZBTB46 expression in ECs in response to d-flow leads to downstream changes in EC gene expression including decreased P21 and SFRP2 gene expression, promoting EC activation, apoptosis, and senescence, and consequently a focal predisposition to atherosclerosis. We address three specific aims for this project: 1) Determine the downstream targets of ZBTB46 involved in flow regulated EC proliferation. First by combining loss of function and gain of function methods and various flow conditions, we will determine if P21 and/or SFRP2 are responsible for the inhibitory effect of ZBTB46 on EC proliferation seen under s-flow. We will also test if P21 and SFRP2 are direct or indirect targets of ZBTB46. Finally, to identify other potential targets of ZBTB46 as a transcription factor, we will use ChIP-seq in ECs expressing ZBTB46, and use the data to test the role of other potential downstream targets of ZBTB46 that could be involved in atherosclerosis in the future. 2) Determine the role of flow regulated ZBTB46 on EC apoptosis, senescence, and inflammation. We will test if EC-ZBTB46 expression affects these aspects of EC function which are seen in atheroprone areas exposed to d-flow, by using siRNA and adenovirus mediated overexpression of ZBTB46 under s-flow and d-flow conditions in conjunction with relevant stimuli such as hydrogen peroxide and TNFa. 3) Determine the effect of EC-ZBTB46 on atherosclerosis in vivo. As ZBTB46 is also expressed in classical dendritic cells which are known to affect atherosclerosis, our team has developed hyperlipidemic EC-targeted ZBTB46-KO mice. Using these mice in experimental models of atherosclerosis, we will determine if loss of ZBTB46 specifically in EC contributes to the flow mediated susceptibility to atherosclerosis by promoting EC apoptosis, senescence, and/or inflammation. Successful completion of our aims will potentially find a novel regulator of EC functions contributing to atherosclerosis and may identify novel potential therapeutic targets for atherosclerosis.